Project 2 Project Summary Marrow infiltrating lymphocytes (MILs) represent an approach to adoptive T cell therapy (ACT) initially developed to enrich for tumor specific T cells from the tumor microenvironment for hematologic malignancies. Our central hypothesis is that intrinsic characteristics of activated tumor-specific marrow infiltrating lymphocytes determine their efficacy. Strategies to augment their anti-tumor efficacy will focus on overcoming their pre-expansion dysfunction and minimizing the induction of tolerance in vivo. These hypotheses will be tested clinically in the context of ongoing or new clinical trials of MILs in patients with relapsed malignancies after alloBMT and those with refractory myeloma as well in preclinical in vitro and murine models. Accordingly, in Specific Aim 1, we will investigate if the efficacy of autoMILs can be augmented by increasing their antigen-specificity or through checkpoint blockade. As a part of this aim we will: i) conduct a pilot randomized phase I/II clinical trial in relapsed multiple myeloma (MM) to evaluate if PD-1 blockade can augment efficacy of autoMILS as ACT in patients with relapsed multiple myeloma; ii) quantify tumor specific immunity and global immune responsiveness to assess overall efficacy. In Specific Aim 2, we will determine the efficacy and mechanisms of activated patient-derived, allogeneic (allo)MILs in augmenting anti-tumor immunity in patients relapsing post-allogeneic BMT. In this aim we will: i) determine the clinical efficacy of alloMILs in patients relapsing after alloBMT and ii) characterize the mechanisms of success and failure in patients with relapsed malignancy after alloBMT treated with alloMILs. Finally, in Specific Aim 3, we will test the hypothesis that MILs anti-tumor activity can be enhanced by overcoming inhibitory checkpoints, harnessing agonists of co-stimulation, and by activating the STING pathway. In aggregate, the proposed research is significant in addressing an important unmet need of reducing or treating relapsed disease following BMT through clinical studies as well as increasing the overall understanding of mechanisms of antitumor immunity and developing innovative ACT strategies utilizing MILs to augment antitumor immunity posttransplant.